食源性诺如病毒单克隆抗体可变区基因克隆及结构特征分析

食源性诺如病毒是引发全球食品安全事件的重要病原,近年来新冠疫情的持续肆虐,更突显了加强食品领域病毒安全研究的紧迫性。该研究以实验室前期获得的食源性诺如病毒高效单克隆抗体为对象,克隆并系统分析了其重链和轻链可变区基因序列。从分泌食源性诺如病毒单克隆抗体的杂交瘤细胞株1E3中提取总RNA,通过RT-PCR扩增单克隆抗体1E3的重链可变区VH和轻链可变区VL的DNA序列。将产物克隆到PMD19-T载体,测序并分析其可变区氨基酸序列。通过NCBIblast比对,显示扩增的VH和VL序列为小鼠抗体可变区序列,进一步利用Vbase2数据库对测序结果进行基因结构分析,定位了VH和VL上的高变区域互补决定区CDR和骨架区域FR,各包含3个CDR和4个FR区域,其中VH片段为360bp,编码120个氨基酸,属于IGHV3-2*02家族;VL片段为339bp,编码113个氨基酸,属于IGKV1-135*01家族。通过分子对接表明抗体重链上位点D108与病毒衣壳P蛋白上N195形成氢键,为关键氨基酸残基。食源性诺如病毒单克隆抗体VH和VL片段的成功扩增促进了基因工程抗体的发展及其在食品安全新型检测与控制技术的应用。

Gene Cloning and Structural Analyses of the Variable Regions of a Monoclonal Antibody against Foodborne Norovirus

Foodborne norovirus is an important pathogen that has triggered food safety incidents worldwide. The continued outbreak of COVID-19 has highlighted the urgency of viral safety research in the food sector. In this study, a highly efficient monoclonal antibody against foodborne norovirus obtained by our team was selected and the variable regions of its heavy chain (VH) and light chain (VL) were cloned and analyzed. Total RNA was extracted from the hybridoma cell line 1E3 secreting monoclonal antibodies against foodborne norovirus, and the DNA sequences of the VH and VL genes of the monoclonal antibody 1E3 were amplified by RT-PCR. The fragments were cloned into the PMD19-T vector and sequenced, and the primary amino acid sequences of both variable regions were then analyzed. An NCBI BLAST comparison confirmed that the amplified VH and VL sequences were of mouse antibody variable regions. Using the VBASE2 database to analyze the variable region-encoding gene structures further, the positions of the three amplified complementarity-determining regions and four amplified framework regions on VH and VL were confirmed to be complete. The VH gene was 360 bp long, encoded 120 amino acids, and belonged to the IGHV3-2*02 family. The VL gene was 339 bp long, encoded 113 amino acids, and belonged to the IGKV1-135*01 family. Through molecular docking experiments, it was found that D108 (the key amino acid residue) of the antibody VH chain bonded to N195 of the viral capsid P protein through hydrogen bonding. The successful amplification of the VH and VL fragments of this monoclonal antibody against foodborne norovirus has promoted the development of genetically engineered antibodies and the application of new microbial detection and control technologies for ensuring food safety.